Throughout this application, various publications are referenced by author and date. Full citations for these publications may be found listed alphabetically at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
Atypical xe2x80x9cantineutrophil cytoplasmic antibodiesxe2x80x9d (ANCA) are present in patients with ulcerative colitis (UC), primary sclerosing cholangitis (PSC), and autoimmune hepatitis (AIH) ANCA represent a family of heterogenous autoantibodies directed against constituents of neutrophilic granulocytes. These autoantibodies have become valuable seromarkers for the diagnostic and therapeutic management of patients with systemic vasculitides such as Wegner granulomatosis and microscopic polyangiitis, in which they recognize well defined cytoplasmic antigens such as proteinase 3 and myeloperoxidase. Two well established ANCA staining patterns can be distinguished on ethanol-fixed neutrophils: a difuse cytoplasmic fluorescence pattern (c-ANCA) and a fine homogeneous labeling of the perinuclear cytoplasm (p-ANCA).
Autoantibodies that are similar to p-ANCA in patients with systemic vasculitides are detected in individuals with chronic inflammatory bowel diseases (IBD) such as ulcerative colitis or autoimmune liver disorders such as primary sclerosing cholangitis (PSC) and autoimmune hepatitis (AIH). Contrary to systemic vasculitides, the role of ANCA in these disorders is not clear. Various cytoplasmic proteins such as bactericidal/permeability increasing protein, catalase, cathepsin G, enolase, or lactoferrin have been proposed as putative target antigens of ANCA in these disorders, but reactivity to these proteins has only been detected in less than thirty five percent of cases. The predominant target antigen of ANCA in IBD and autoimmune liver disorders has not been identified. Since their target antigens are unknown, p-ANCA in patients with IBD or autoimmune liver disorders are generally referred to as atypical p-ANCA.
ANCA are generally defined as autoantibodies directed against cytoplasmic antigens localized in the azurophil and specific granules of neutrophils (Woude et al., 1985; Savige et al, 1999). Using double-labeling immunofluorescence microscopy, it was shown that this definition holds true only for C-ANCA and classic p-ANCA in systemic vasculitides, but not for ANCA in individuals with IBD and autoimmune liver disease (Billing et al, 1995; Terjung et al., 1998). ANCA in those disorders do not react with cytoplasmic structures (Billing et al, 1995; Terjung et al., 1998). Their fluorescence pattern examined by indirect immunofluorescence microscopy is characterized by a broad inhomogeneous labeling of the nuclear periphery along with multiple intranuclear fluorescent foci. By means of immuno electron microscopy, it has been shown that this focal intranuclear fluorescence likely corresponds to invaginations of the nuclear envelope (Fricker et al., 1997; Terjung et al., 1998). These labeling characteristics of atypical p-ANCA in IBD and autoimmune liver disorders strongly suggest that a nuclear antigen is the target (possibly the term ANCA (antineutrophil cytoplasmic antibody) is a misnomer, and the term ANNA (antineutrophil nuclear antibody) is more appropriate).
Previously, it has been suggested that histone H1, particularly its isoform H1-3, is a candidate antigen recognized by atypical p-ANCA in patients with UC (Targan et al., 1996; Cohavy et al., 1997,; and Cohavy et al. 1998). Furthermore, reactivity to this nuclear antigen in 42 percent of sera from T-cell receptor alpha-deficient mice that developed UC like syndrome and whose sera contained ANCA has also been reported (Mizoguchi et al., 1997). In addition, eighty nine percent of sampled patients with AIH had antibodies that recognized high mobility group (HMG xc2xd) non-histone chromosomal proteins (Sobajima et al., 1997). Although neither histone H1 nor HMG xc2xd proteins are neutrophil specific, it has been hypothesized that certain epitopes of these nuclear proteins are only immunoaccessible in neutrophils and therefore might represent target proteins of atypical p-ANCA (Targan et al., 1996; Cohavy et al., 1997,; and Cohavy et al. 1998). However, these studies do not exclude the possibility that atypical p-ANCA predominantly recognize another myeloid specific nuclear protein.
Therefore, a need arises to characterize the nuclear antigen recognized by atypical p-ANCA. The main impetus behind this need is that molecular identification of the target antigen will result in the development of highly specific, sensitive, and reproducible assays for the detection of atypical p-ANCA in the routine clinical laboratory setting. These assays will have diagnostic use for patients with IBD, in particular UC and autoimmune liver disorders, in particular PSC ans AIH.
The present invention is directed to the molecular characterization of the nuclear antigen recognized by atypical p-antineutrophil cytoplasmic antibodies (p-ANCA), thus permitting better diagnosis of patients with inflammatory bowel diseases such as ulcerative colitis (UC), and autoimmune liver diseases such as primary sclerosing cholangitis (PSC), and autoimmune hepatitis (AIH). Molecular characterization of the target antigen comprises preparing cytoplasmic and nuclear extracts of human neutrophils, human HL-60 and murine 32D myeloid cells. Proteins are resolved by 1 and 2 dimensional gel electrophoresis and reactive proteins are detected by immunoblotting with sera from individuals, making certain to have both normal and disease controls. Atypical p-ANCA can further be affinity purified against the reactive protein and investigated for their immunofluorescence pattern using confocal microscopy. The antigen that atypical p-ANCA can recognize is isolated or partially purified and used to detect the presence of atypical p-antineutrophil cytoplasmic antibodies so as to diagnose patients with inflammatory bowel diseases such as ulcerative colitis (UC), and autoimmune liver disease such as primary sclerosing cholangitis (PSC) and autoimmune hepatitis (AIH).